How and why to study autophagy in Drosophila: it's more than just a garbage chute.

Nagy P, Varga Á, Kovács AL, Takáts S, Juhász G - Methods (2014)

Bottom Line:
During the catabolic process of autophagy, cytoplasmic material is transported to the lysosome for degradation and recycling.This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.

f0015: Specific markers of autophagy. (A) The most popular markers of autophagy are fluorescently tagged Atg8a reporters, such as mCherry-Atg8a. Atg8a is delivered to autolysosomes, where mCherry retains its fluorescence and accumulates to high levels. Thus, autolysosomes are labeled bright red in GFP-negative control cells, whereas autophagosomes appear fainter and smaller, as seen in the two GFP-positive cells expressing Syntaxin 17 RNAi, which silences the SNARE required for autophagosome-lysosome fusion. (B) Inhibition of Tor kinase increases autophagic flux, resulting in enhanced delivery of mCherry-Atg8a to lysosomes. Thus, autolysosomes are labeled much brighter with mCherry in GFP-positive cells in which Raptor (encoding an essential subunit of Tor kinase complex 1) is knocked down than in surrounding control cells. Note that mCherry signal in control cells looks fainter than in panel A because this image was taken using a much shorter exposure time. (C) and (D) Atg1 RNAi in GFP-positive cells inhibits puncta formation of both mCherry-Atg8a (C) and mCherry-Atg18 (D), as Atg1 acts upstream of Atg8a and Atg18 in the hierarchy of Atg proteins. (E) Knockdown of Atg12 blocks the generation of mCherry-Atg8a dots. (F) Small mCherry-Atg18 dots likely representing stalled phagophore assembly sites (PASs) accumulate in GFP-positive cells undergoing Atg12 RNAi, as Atg18 acts upstream of Atg12. (G) and (H) Protein aggregates containing p62 (green) accumulate in large numbers in the brain of Atg7 mutant flies compared to controls. Note that association of the upstream-acting Atg1 kinase subunit FIP200 (red) with p62 is also increased in Atg7 mutants, suggesting that such aggregates might represent stalled phagophore assembly sites. (I) Phagophores that are positive for endogenous Atg8a associate with large protein aggregates formed upon high-level overexpression of GFP-p62. (J) Ultrastructural analysis demonstrates an enlarged phagophore (P) attached to the surface of a protein aggregate in a fat body cell of a starved larva as in panel I. ap: autophagosome; m: mitochondrion. Panels A–F, I, J show fat body cells of starved L3 stage larvae, and panels G, H show adult brains. Bars equal 20 μm in panels A (for A–F), G, H. Bar in I equals 4 μm, and bar in J represents 1 μm, respectively.

Mentions:
It is worth mentioning that there are two paralogs of yeast Atg8 in Drosophila. Atg8a is highly expressed in all tissues, whereas Atg8b only shows strong expression in the testis [37]. Thus, most assays rely on tagged Atg8a. A particularly popular marker for studying autophagy in Drosophila is mCherry-Atg8a, because it allows the visualization of all autophagic structures: phagophores, autophagosomes and autolysosomes [38]. This is because Atg8 family proteins are bound to both the inner and outer membranes of autophagosomes, so half of these molecules are delivered to the lysosome in each autophagosomal cycle. Likewise, fluorescently tagged Atg8a also gets into the acidic lysosomal lumen, where GFP is quenched rather quickly but mCherry retains its fluorescence much longer. This is due to their different pKa values [39]. Thus, GFP-Atg8a labels phagophores and autophagosomes more specifically, and only a subset of autolysosomes may be positive for this reporter [6,10,21,30]. In contrast, mCherry-Atg8a accumulates to high levels in autolysosomes, so phagophores and autophagosomes appear fainter (Fig. 3A and B). It is worth noting that the intensity of the mCherry signal may be used to estimate the rate of autophagic protein delivery to lysosomes in these experiments, as a block of autophagosome-lysosome fusion by knocking down the autophagosomal SNARE Syntaxin 17 prevents the formation of highly fluorescent autolysosomes (Fig. 3A) [40], whereas enhanced autophagy due to Tor kinase inhibition results in extremely high levels of mCherry in autolysosomes. As a consequence, these appear much brighter in Tor inactivated cells than the structures seen in surrounding control cells (Fig. 3B). However, sometimes there may be problems with the specificity of overexpressed Atg8 reporters. For example, high-level expression of Atg8a was found to rescue the autophagy-inhibiting phenotype of dominant-negative Atg4 [41]. More importantly, overexpressed Atg8a reporter molecules are captured into the large protein aggregates that form in fat body cells for example during proteasome inhibition [42]. These Atg8a-positive structures may as well be falsely interpreted as large autophagic vesicles, but ultrastructural analysis clearly showed that they are in fact cytosolic protein aggregates. In addition, protein aggregates in proteasome RNAi cells did not stain positive for endogenous Atg8a [42]. Except this unusual situation, indirect immunofluorescence analysis of Atg8a using a specific antibody should give a labeling similar to GFP-Atg8a. One advantage of this technique is that it is based on following the endogenous protein instead of an overexpressed tagged reporter. It is also much faster to carry out, as one does not need to cross the reporter into the genetic background of interest. Several anti-Atg8a antibodies have been published that work well for microscopy in Drosophila[16,40,43,44]. Of note, a GFP-Atg8a reporter expressed from the genomic promoter of Atg8a has also been published, which can be used to visualize autophagosomes in the midgut [45]. Unfortunately its expression level is relatively low, and we could not detect it in the larval fat body (our unpublished results). Alternatively, a heat shock-inducible GFP-Atg8a transgene may be used in a sort of pulse-chase experiment to identify autophagosomes and follow their degradation over time [6].

f0015: Specific markers of autophagy. (A) The most popular markers of autophagy are fluorescently tagged Atg8a reporters, such as mCherry-Atg8a. Atg8a is delivered to autolysosomes, where mCherry retains its fluorescence and accumulates to high levels. Thus, autolysosomes are labeled bright red in GFP-negative control cells, whereas autophagosomes appear fainter and smaller, as seen in the two GFP-positive cells expressing Syntaxin 17 RNAi, which silences the SNARE required for autophagosome-lysosome fusion. (B) Inhibition of Tor kinase increases autophagic flux, resulting in enhanced delivery of mCherry-Atg8a to lysosomes. Thus, autolysosomes are labeled much brighter with mCherry in GFP-positive cells in which Raptor (encoding an essential subunit of Tor kinase complex 1) is knocked down than in surrounding control cells. Note that mCherry signal in control cells looks fainter than in panel A because this image was taken using a much shorter exposure time. (C) and (D) Atg1 RNAi in GFP-positive cells inhibits puncta formation of both mCherry-Atg8a (C) and mCherry-Atg18 (D), as Atg1 acts upstream of Atg8a and Atg18 in the hierarchy of Atg proteins. (E) Knockdown of Atg12 blocks the generation of mCherry-Atg8a dots. (F) Small mCherry-Atg18 dots likely representing stalled phagophore assembly sites (PASs) accumulate in GFP-positive cells undergoing Atg12 RNAi, as Atg18 acts upstream of Atg12. (G) and (H) Protein aggregates containing p62 (green) accumulate in large numbers in the brain of Atg7 mutant flies compared to controls. Note that association of the upstream-acting Atg1 kinase subunit FIP200 (red) with p62 is also increased in Atg7 mutants, suggesting that such aggregates might represent stalled phagophore assembly sites. (I) Phagophores that are positive for endogenous Atg8a associate with large protein aggregates formed upon high-level overexpression of GFP-p62. (J) Ultrastructural analysis demonstrates an enlarged phagophore (P) attached to the surface of a protein aggregate in a fat body cell of a starved larva as in panel I. ap: autophagosome; m: mitochondrion. Panels A–F, I, J show fat body cells of starved L3 stage larvae, and panels G, H show adult brains. Bars equal 20 μm in panels A (for A–F), G, H. Bar in I equals 4 μm, and bar in J represents 1 μm, respectively.

Mentions:
It is worth mentioning that there are two paralogs of yeast Atg8 in Drosophila. Atg8a is highly expressed in all tissues, whereas Atg8b only shows strong expression in the testis [37]. Thus, most assays rely on tagged Atg8a. A particularly popular marker for studying autophagy in Drosophila is mCherry-Atg8a, because it allows the visualization of all autophagic structures: phagophores, autophagosomes and autolysosomes [38]. This is because Atg8 family proteins are bound to both the inner and outer membranes of autophagosomes, so half of these molecules are delivered to the lysosome in each autophagosomal cycle. Likewise, fluorescently tagged Atg8a also gets into the acidic lysosomal lumen, where GFP is quenched rather quickly but mCherry retains its fluorescence much longer. This is due to their different pKa values [39]. Thus, GFP-Atg8a labels phagophores and autophagosomes more specifically, and only a subset of autolysosomes may be positive for this reporter [6,10,21,30]. In contrast, mCherry-Atg8a accumulates to high levels in autolysosomes, so phagophores and autophagosomes appear fainter (Fig. 3A and B). It is worth noting that the intensity of the mCherry signal may be used to estimate the rate of autophagic protein delivery to lysosomes in these experiments, as a block of autophagosome-lysosome fusion by knocking down the autophagosomal SNARE Syntaxin 17 prevents the formation of highly fluorescent autolysosomes (Fig. 3A) [40], whereas enhanced autophagy due to Tor kinase inhibition results in extremely high levels of mCherry in autolysosomes. As a consequence, these appear much brighter in Tor inactivated cells than the structures seen in surrounding control cells (Fig. 3B). However, sometimes there may be problems with the specificity of overexpressed Atg8 reporters. For example, high-level expression of Atg8a was found to rescue the autophagy-inhibiting phenotype of dominant-negative Atg4 [41]. More importantly, overexpressed Atg8a reporter molecules are captured into the large protein aggregates that form in fat body cells for example during proteasome inhibition [42]. These Atg8a-positive structures may as well be falsely interpreted as large autophagic vesicles, but ultrastructural analysis clearly showed that they are in fact cytosolic protein aggregates. In addition, protein aggregates in proteasome RNAi cells did not stain positive for endogenous Atg8a [42]. Except this unusual situation, indirect immunofluorescence analysis of Atg8a using a specific antibody should give a labeling similar to GFP-Atg8a. One advantage of this technique is that it is based on following the endogenous protein instead of an overexpressed tagged reporter. It is also much faster to carry out, as one does not need to cross the reporter into the genetic background of interest. Several anti-Atg8a antibodies have been published that work well for microscopy in Drosophila[16,40,43,44]. Of note, a GFP-Atg8a reporter expressed from the genomic promoter of Atg8a has also been published, which can be used to visualize autophagosomes in the midgut [45]. Unfortunately its expression level is relatively low, and we could not detect it in the larval fat body (our unpublished results). Alternatively, a heat shock-inducible GFP-Atg8a transgene may be used in a sort of pulse-chase experiment to identify autophagosomes and follow their degradation over time [6].

Bottom Line:
During the catabolic process of autophagy, cytoplasmic material is transported to the lysosome for degradation and recycling.This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.